1. Field of the Invention
The invention relates to a method for determining the binding behavior of ligands which specifically bind to target molecules at at least one binding site.
2. Discussion of Background Information
DE 198 14 775 A1 discloses a method for determining the binding constants of dissolved agents and substances on surfaces constituted by amphiphilic molecules. Therein, layers of amphiphilic molecules are bonded to a solid carrier so that a solid-supported membrane is formed. A defined quantity of this solid-supported membrane is contacted with a mobile phase. The mobile phase contains a defined quantity of substances which are to be examined with respect to lipid binding. After an incubation, the solid-supported membranes are separated from the mobile phase. The concentrations of the substances in the mobile phase or the solid-supported membrane are determined. The method can be carried out with solid-supported membranes and substances that are present in different quantity ratios. From the determined concentrations, the binding constant can be calculated.
From WO 99/50669 A1 there is known a method for screening a compound library to determine the relative affinity of a plurality of putative ligands to a target receptor in relation to an indicator compound by means of frontal chromatography. Therein, the target receptors are immobilized on the solid phase of a chromatographic column. The compound library is applied onto the chromatographic column and equilibrated therewith at least partially. Subsequently, the indicator compound is applied and its break through time, i.e., the time until it appears in the effluent of the chromatographic column, is determined by means of mass spectrometry. The break through time of the indicator compound is affected by its affinity, the affinity of ligands present to the target receptor, and the number of target receptors which are not occupied by the ligands. From the change in the break through time in comparison to the known break through time of the indicator compound, it can be determined whether compounds of the compound library have an affinity to the target receptors, and whether this affinity is higher or lower than that of the indicator compound.
A disadvantage of this method is that the target receptors are invariably immobilized. They are not present in the native state, whereby it is possible to only a limited extent to say something about the binding behavior on native target receptors. A further disadvantage of the method is that in kinetic studies, it leads to incorrect results regarding the binding behavior between ligands and the indicator compound. If a ligand and the indicator compound had the same affinities to the target receptor, the affinity of the ligand would be determined as too low if the ligand shows slower binding kinetics relative to the indicator compound. A further disadvantage is that the quantity of the indicator compound which is bound or unbound, respectively, in the binding equilibrium state cannot be determined.
WO 01/22078 A1 relates to a method for the identification of an active chemical substance from a mixture of active and inactive chemical substances, where a target substance is added to the mixture. Complexes formed of the active substance and the target substance are separated from the mixture. For identification, the active chemical substance may be released from the complexes and identified by, for example, mass spectrometry. Alternatively, the active substance may also be identified by comparing a chromatogram or a mass spectrum, respectively, of each of the mixture of active and inactive substances and the mixture after separation of the complex. A disadvantage thereof is that it must be possible to determine each of the substances of the mixture.
WO 97/43301 A2 relates to a method for characterizing the members of a combinatorial library which bind to a certain domain. Therein, the domain is mixed with the members of the combinatorial library to permit a binding of the members to the domain. The formed complexes are separated from the unbound members. The bound members are eluted from the complexes and analyzed by mass spectrometry. Therein, it is possible to elute more weakly bound members separately from more strongly bound members. A disadvantage thereof is that each of the members of the mixture must be determinable by mass spectrometry.
From WO 96/22530 A1 and U.S. Pat. No. 5,891,742 A there is known a method for determining a compound which binds to a target compound from a mixture of compounds. Therein, the target compound is contacted with the mixture of compounds. Unbound compounds are separated from formed complexes of the compounds with the target molecules. The formed complexes are analyzed by mass spectrometry. It is of disadvantage therein that all compounds and complexes, respectively, of the binding assay must be determinable by mass spectrometry.
From WO 00/00823 A1 there is known a method for identifying a ligand which covalently binds to a target molecule. In this method, a target molecule is contacted with several compounds of a substance library. The compound which forms a covalent bond with a chemically reactive group of the target molecule is identified by mass spectrometry. For this purpose, the conjugate formed by the covalent bond may be fragmented. The bound compound may as well be released by cleaving a disulfide bond. For identification, the mass spectroscopic characteristics of the compounds to be determined or of the fragment to be determined, respectively, must be known in this method.
From WO 99/45150 A1 there is known a method for determining the relative binding affinity to a target substance of compounds of a combinatorial mixture. Therein, a first complex of the target substance and a standard compound binding thereto is contacted with the combinatorial mixture of compounds, whereby second complexes are formed from these compounds and the target substance. The mixture is analyzed by mass spectrometry. From the quantity of first and second complexes contained therein, the relative binding affinity can be determined. Also in this assay, it must be possible to analyze all compounds which potentially form complexes with the target molecules, and the complexes formed therefrom, respectively.
From WO 00/47999 A1 there is known a method for screening complex biological samples for a ligand which binds to a target protein. Therein, the target protein is mixed with the complex biological sample and incubated under conditions which permit the formation of complexes of ligands with the target protein. Formed ligand-target protein complexes are separated and caused to dissociate. Released ligands can be analyzed by mass spectrometry following the separation of the target protein. The separation is effected in each case by means of size exclusion processes. Furthermore, WO 00/47999 A1 discloses the use of a reference substance and of a known competitive ligand. The competitive ligand serves to determine whether a ligand binds specifically or non-specifically to the selected target protein. For this purpose, the competitive ligand is mixed with the complex biological sample, the process is carried out as described, and it is determined whether the mass spectroscopic signal of the ligand is affected by the presence of the competitive ligand. The method discloses a comparison of the binding assays in the presence and absence of the competitive ligand. However, all bound ligands are invariably determined by mass spectrometry.
Moreover, it is generally known form the prior art to indirectly determine the binding of unlabeled ligands to a target molecule by means of ligands which are labeled with a marker substance. Therein, the unlabeled ligands are incubated with the target molecules together with a defined portion of labeled ligands. The subsequent determination of the quantity of the marker substance which is bound to the target molecules through the labeled ligands allows the determination of the binding behavior of the ligands which are bound to the target molecules.
The methods known in the prior art make it necessary to directly or indirectly quantify either the bound or the unbound ligands. With direct quantification, this may involve a substantial expenditure, depending on the ligand. For quantification, the employed detection system must be calibrated for each of the ligands to be quantified. This is necessary also for ligands of which it is not known whether they show an affinity to the target molecules. The indirect quantification by means of labeled ligands involves substantial expenditure as well. Initially it is necessary to label the ligands with a marker substance so that the labeled ligands still have sufficient affinity to the target molecules. Frequently, various labeling methods with various marker substances must be tested for this purpose. Often, only radioactive marker substances prove to be suitable. Handling these marker substances and the ligands labeled therewith is dangerous, and because of the required safety precautions, it involves substantial expenditure. Furthermore, not every ligand is suitable for labeling by a certain desired method. This may necessitate a search for suitable ligands.
Object of the present invention is the elimination of the disadvantages of the prior art. In particular, there is to be provided a method which makes it possible to determine, with little expenditure, the specific binding behavior of any ligands to any desired target molecules, unaffected to a large extent by the kinetics of the binding of the ligands to the target molecules.
This problem is solved by the features of the claimed invention.